Studies of catechol-O-methyltransferase (COMT), an enzyme important in the extraneuronal inactivation of catecholamines (dopamine, norepinephrine, and epinephrine) and the detoxification of xenobiotic catechols, will be undertaken in an effort to obtain the information necessary for the rational design of COMT inhibitors. Studies will include the use of affinity labeling reagents to identify the nucleophilic groups present at the active site of this enzyme. Several classes of affinity labeling reagents have already been identified in our laboratory and have been shown to selectively modify different nucleophilic residues at the COMT active site. These reagents provide the tools necessary to label these amino acid residues so they can be isolated and identified. Valuable information about the active site of COMT and the mechanism by which COMT catalyzes its transmethylation should be obtained from these studies. In addition these affinity labeling reagents will be utilized in vivo for the subcellular localization of this enzyme in rabbit thoracic aorta. The synthesis of a series of 5-substituted-3-hydroxy-4-methoxyphenylethylamines and 5-substituted-3-hydroxy-4-methoxybenzoic acids will also be undertaken in an attempt to develop effective in vivo inhibitors of COMT. The potential inhibitors will be evaluated in vitro for their inhibitory effects on COMT and phenol sulfotransferase and also for their activity as substrates for the latter enzyme system. The effectiveness of these compounds as inhibitors of O-methylation in vivo will be compared with their activity in vitro as inhibitors of COMT and as substrates for phenol sulfotransferase. Such information should lead to the development of inhibitors of COMT which will be effective as chemotherapeutic agents in vivo. Such inhibitors of COMT could be utilized to control the metabolism of endogenous catecholamines or to alter the metabolism of exogenously administered catecholic drugs.